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Keywords:

  • Ketogenic diet;
  • Polyunsaturated fatty acids;
  • Ketones;
  • Acetoacetate;
  • Brain;
  • Positron emission tomography;
  • Epilepsy

Summary

  1. Top of page
  2. Plasma PUFA in Children on the KD
  3. PUFA Metabolism in Rats on the KD
  4. Brain PET Studies with 11C-AcAc
  5. Conclusion
  6. Acknowledgment
  7. References

Changes in the metabolism of polyunsaturated fatty acids (PUFA), both in children on the high fat ketogenic diet (KD) for seizure control and in rats on a KD enriched in PUFA, raise the possibility that increased brain arachidonic acid (ARA) and/or docosahexaenoic acid (DHA) may contribute to better seizure control. Our studies with PUFA and several other reports raise the question of whether persistent ketonemia or elevated brain uptake of ketones are strictly necessary for the clinical effectiveness of the KD in intractable epilepsy. To address this question, we have developed the synthesis of carbon-11 labeled acetoacetate (11C-AcAc) for PET studies to investigate brain ketone uptake directly in humans and animals. In rats on the KD for 10 days, 11C-AcAc uptake by the brain increased 7- to 8-fold, an increase similar to that induced by 48 h fasting. In rats and humans, paired PET scans (11C-AcAc followed immediately by18fluorodeoxyglucose) will be conducted to assess the uptake of AcAc and glucose by the brain while on the KD and in neurological disorders associated with aging.

Despite its lengthy clinical use, the mechanism by which the very high-fat ketogenic diet (KD) reduces epileptic seizures in children or animal models is still unknown. Even the role of elevated plasma ketones is unclear. Here, we first briefly describe our contributions in this field concerning the possible link between polyunsaturated fatty acids (PUFA) and efficacy of the KD, and second, the application of positron emission tomography (PET) to imaging brain ketone (acetoacetate; AcAc) uptake in rats.

The membrane phospholipids of neural cells naturally contain large amounts of PUFA, principally arachidonic acid (ARA, 20:4ω6), and docosahexaenoic acid (DHA, 22:6ω3). We observe a bidirectional interaction between PUFA intake or metabolism and ketosis: on the one hand, raising the proportion of PUFA in the KD raises plasma β-hydroxybutyrate (β-HBA) in rats. On the other hand, independent of its PUFA content, the KD has a marked impact on plasma PUFA in children and on plasma and tissue PUFA content and metabolism in rats. Indeed, the paradox of transitory induction of ketosis, yet persistent and marked changes in PUFA metabolism while on the KD, brings to the fore the importance of being able to assess brain uptake and/or levels of ketones while on the KD. Hence, we have recently synthesized carbon-11 (11C)-AcAc so as to be able to utilize positron tomography (PET) to address this question directly.

Plasma PUFA in Children on the KD

  1. Top of page
  2. Plasma PUFA in Children on the KD
  3. PUFA Metabolism in Rats on the KD
  4. Brain PET Studies with 11C-AcAc
  5. Conclusion
  6. Acknowledgment
  7. References

We have reported that the KD significantly raises the PUFA content of plasma lipids in children with refractory epilepsy (Fraser et al., 2003). The children (aged 6 ± 5 years; mean ± SD) had blood samples taken before and 3–4 weeks after starting the KD. The KD raised fasting plasma β-HBA from 0.5 ± 0.2 to 3.8 ± 1.6 mM, and reduced seizures by 50–100% in 78% of the children. While on the KD, plasma total free fatty acids were increased 2.2-fold. Depending on the plasma lipid class, ARA and DHA were 1.6- to 2.9-fold higher on the KD. These results are consistent with the possibility that seizure protection while on the KD is achieved in part via increased availability of certain PUFA to the brain, notably DHA and/or ARA (Voskuyl et al., 1998).

PUFA Metabolism in Rats on the KD

  1. Top of page
  2. Plasma PUFA in Children on the KD
  3. PUFA Metabolism in Rats on the KD
  4. Brain PET Studies with 11C-AcAc
  5. Conclusion
  6. Acknowledgment
  7. References

To further assess changes in PUFA metabolism while on the KD, we examined tissue PUFA levels and metabolism of carbon-13 labeled α-linolenic acid (13C-ALA; 18:3ω3) in rats on the classic 4:1 KD (Taha et al., 2005). As we and others (Likhodii et al., 2000; Taha et al., 2005) have observed, the initial increase in plasma β-HBA was not sustained; rather, β-HBA increased transiently from 0.4 ± 0.2 to 0.8 ± 0.6 mM but returned to starting values within 10 days. Despite the loss of ketonemia in the rats, fatty acid metabolism was markedly affected including a reduced percent of 18–22 carbon PUFA in adipose tissue and plasma. Hence, as in children (Fraser et al., 2003), the KD significantly changed tissue distribution of PUFA (Fig. 1), but in the rat, plasma PUFA were decreased (Taha et al., 2005) whereas in the children, they were increased (Fraser et al., 2003). Nevertheless, the effect implicated ARA and DHA in particular, raising brain ARA and DHA by ∼15% in the rats. Interestingly, without increasing ARA or DHA intake in the KD for the rat study, both were raised in brain (Taha et al., 2005). These results in the rat model (Taha et al., 2005) were observed in the absence of a difference in plasma β-HBA between the control and KD groups, which led us to ask whether persistent ketonemia or, indeed, elevated brain uptake of ketones are necessary for the clinical effectiveness of the KD.

image

Figure 1. Overview of the changes in metabolism of polyunsaturated fatty acids on the ketogenic diet (KD) in rats. ALA, α-linolenic acid; ARA, arachidonic acid; DHA, docosahexaenoic acid; FFA, free fatty acids; LA, linoleic acid; TG, triglycerides.

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Brain PET Studies with 11C-AcAc

  1. Top of page
  2. Plasma PUFA in Children on the KD
  3. PUFA Metabolism in Rats on the KD
  4. Brain PET Studies with 11C-AcAc
  5. Conclusion
  6. Acknowledgment
  7. References

Direct (Blomqvist et al., 2002, 1995) and indirect (Hasselbalch et al., 1996) studies suggest that brain ketone uptake in humans is proportional to and responds rapidly to a rise in plasma ketones. To further investigate brain ketone metabolism in both humans and animals, we have developed the synthesis of 11C-AcAc and 11C-β-HBA (Tremblay et al., 2007, 2008) for human and animal PET studies (Fig. 2). In our first studies with rats on the KD for 10 days, brain 11C-AcAc uptake increased 7- to 8-fold, an increase equivalent to that induced by 48 h fasting (Fig. 3) (Bentourkia et al., 2006).

image

Figure 2. Autoradiograph of 11C-acetoacetate uptake in the rat brain (A) with corresponding magnetic resonance image of the same cross-section of rat brain (B). 10 minutes after injecting 11C-acetoacetate into the rat's caudal vein under anesthesia, the chest was opened and brain fixed by intracardiac perfusion of 4% formalin. The brain was dissected and slices exposed in a filmless cassette for at least 2 h and revealed on a phosphoimager.

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image

Figure 3. Brain uptake of 11C-acetoacetate determined by positron emission tomography in rats given a control (5 wt% fat) or a ketogenic diet (90 wt% fat) for 10 days, or fasted for 48 h (n = 4 per group; mean ± SD; 11). Control values are normalized to 100%. Values in the ketotic or fasted rats exceeded those in controls by 7.2- and 8.1-fold, respectively (p  <  0.01).

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We plan to do paired PET scans with 11C-AcAc followed immediately by 18fluorodeoxyglucose, which should help answer questions concerning the uptake of both the brain's major fuels while on the KD. One such question is whether the KD alters brain glucose uptake; humans studies suggest not (Taha et al., 2005) while a recent animal study concluded that the KD may raise brain glucose uptake (Puchowicz et al., 2007). Given the acute seizure sensitivity of children on the KD to dietary glucose, this question is of particular importance both clinically and for understanding the mechanism of the KD for seizure control. Brain PET studies with 11C-AcAc not only have application in studies of intractable epilepsy but also in other neurological disorders including cognitive decline in the elderly (Freemantle et al., 2008).

Conclusion

  1. Top of page
  2. Plasma PUFA in Children on the KD
  3. PUFA Metabolism in Rats on the KD
  4. Brain PET Studies with 11C-AcAc
  5. Conclusion
  6. Acknowledgment
  7. References

The beneficial effect of the KD to control intractable seizures is clinically well established but the mechanism remains elusive. As active components of intermediary metabolism, elevated ketones undoubtedly have several effects on brain function, including roles as excellent replacement fuels for glucose and substrates for many different pathways including those of neurotransmitter and lipid synthesis. Therefore, the changes in PUFA metabolism on the KD should not be surprising, but the rise in brain DHA and ARA in rats on the KD (Taha et al., 2005) is both novel and potentially clinically important. The fact that raising intake of the PUFA, ALA, increases ketosis in rats on the KD (Likhodii et al., 2000) is also novel and consistent with extensive recycling of ALA carbon into lipid synthesis (Cunnane et al., 2003). With the availability of 11C-AcAc for human and animal PET studies, connecting the clinical effects of the KD to possible changes in brain ketone uptake can now be explored in vivo and in humans as well as in animal models.

Acknowledgment

  1. Top of page
  2. Plasma PUFA in Children on the KD
  3. PUFA Metabolism in Rats on the KD
  4. Brain PET Studies with 11C-AcAc
  5. Conclusion
  6. Acknowledgment
  7. References

We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Financial support was provided by the Canada Research Chairs (SCC), Department of Medicine fellowship, Université de Sherbrooke (MP, FP), Canadian Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, Canadian Institutes of Health Research, Fonds de recherche en Santé du Québec (MP) and the Research Center on Aging.

Disclosure: The authors declare no conflicts of interest.

References

  1. Top of page
  2. Plasma PUFA in Children on the KD
  3. PUFA Metabolism in Rats on the KD
  4. Brain PET Studies with 11C-AcAc
  5. Conclusion
  6. Acknowledgment
  7. References
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